Triple-negative breast cancers (TNBCs), which do not express estrogen receptor or progesterone receptor or overexpress human epidermal growth factor-related 2 (HER2), are clinically aggressive tumors characterized by high rates of metastases and poor prognosis. Recurrent TNBC is thought to be mediated by a subpopulation of tumor-initiating cells that escape the cytotoxic effects of chemotherapy and are characterized by cell surface expression of CD44. The high incidence of CD44+ cells in TNBC cell lines, many of which also have constitutive activation of the transcription factor STAT3, suggests that CD44 is not merely a marker of tumor-initiating cells, as it is generally viewed, but rather directly involved in modulating STAT3 activity and contributing to the inappropriate survival of these cells. We have demonstrated that CD44 knockdown decreases STAT3 transcriptional activity but not STAT3 tyrosine or serine phosphorylation in TNBC cells. CD44 knockdown also increases TNBC cell sensitivity to conventional chemotherapeutic agents, supporting the importance of CD44 in the pathogenesis of these tumors. However, the molecular mechanism by which CD44 regulates STAT3 activity in TNBC is not yet understood. The goals of this research proposal are: (1) To elucidate the signaling pathway by which CD44 modulates STAT3 transcriptional activity in TNBC, (2) To determine the function of CD44 in STAT3-mediated gene regulation in TNBC, using chromatin immunoprecipitation to analyze the role of CD44 in defining STAT3 genomic binding sites and using mass spectrometry to identify the function of CD44 on STAT3-associated proteins, and (3) To dissect the effect of STAT3 inhibition on the biology of CD44+ versus CD44- TNBC subpopulations, using BH3 profiling to measure cell priming for apoptosis. The completion of this proposal will provide insights into the role of the CD44-STAT3 axis in the pathogenesis of TNBC whose aggressive nature presents serious obstacles to effective treatment. Elucidating how CD44 expression contributes to the aggressive phenotype of TNBC will expand our knowledge of STAT3 biology and may suggest novel therapeutic approaches for tumors with high CD44 expression. Furthermore, increased understanding of the CD44 signaling pathway may ultimately provide clues about how other cancer surface markers are involved in tumorigenesis.